Composition for preventing, ameliorating or treating cognitive impairment and Alzheimer's disease, comprising Lactobacillus fermentum SRK414 strain

Abstract

The present invention relates to a composition for preventing, ameliorating, or treating cognitive impairment or Alzheimer's disease, comprising the Lactobacillus fermentum SRK414 strain (accession number KCTC13687BP) as an active ingredient. The strain of the present invention has the effect of reducing barrier permeability, is highly effective in reducing amyloid beta and tau protein, and is highly effective in improving cognitive function, and can therefore be useful as a food or therapeutic agent for the same purposes.

Description

Detailed Description of the Invention

[0001] [Technical field] This patent application claims priority to Korean Patent Application No. 10-2022-0144820 filed with the Korean Intellectual Property Office on November 2, 2022, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a composition for preventing, ameliorating or treating cognitive impairment and Alzheimer's disease, which comprises the Lactobacillus fermentum SRK414 strain.

[0003] [Background technology] Alzheimer's disease (AD) is a degenerative brain disorder that is one of the most common forms of dementia. Patients with AD experience initial memory loss, followed by a gradual decline in various cognitive functions, including language and judgment, as the disease progresses, ultimately causing serious problems in daily life. While the exact cause and mechanism of Alzheimer's disease are still unknown, it is believed to be primarily caused by neuronal damage due to the abnormal accumulation of amyloid beta protein (Aβ) and tau protein (Tau) in brain tissue, which in turn leads to excessive activation of immune cells in the brain. Because AD is a typical brain disease caused by these major hypotheses, most currently available treatments address problems with signal transmission between brain neurons, and research for therapeutic drug development has primarily been limited to the brain.

[0004] Numerous research findings have revealed that gut microbes play a crucial role in the bidirectional interactions between the gut and the brain, mediated by the endocrine, nervous, and immune systems. Detailed mechanisms have been identified by which changes in gut microbial composition and metabolites directly affect brain functions such as appetite, sleep, and mood regulation, as well as memory and learning, through the mediation of systemic immunity, hormones, and neurotransmitters. As the correlation between the gut and the brain becomes clearer, a growing number of studies have confirmed the role of gut microbes in various brain diseases, sometimes referred to as the gut-brain axis. Based on this gut-brain axis theory, active research is currently underway to identify candidate microbes for the treatment of degenerative brain diseases such as Parkinson's disease and AD using gut microbes.

[0005] [Summary of the Invention] [Problem to be solved by the invention] The present inventors have conducted extensive research to develop a substance that utilizes intestinal microorganisms to prevent, improve, or treat cognitive impairment and Alzheimer's disease. As a result, they discovered that Lactobacillus fermentum SRK414 improves barrier permeability, reduces amyloid beta and tau proteins, which are indicators of degenerative brain diseases such as Alzheimer's, and improves cognitive function in animal models, leading to the completion of the present invention.

[0006] Therefore, an object of the present invention is to provide a composition for preventing, ameliorating or treating cognitive impairment or Alzheimer's disease, which comprises the Lactobacillus fermentum SRK414 strain, accession number KCTC13687BP, as an active ingredient.

[0007] Another object of the present invention relates to the use of the Lactobacillus fermentum SRK414 strain deposited under accession number KCTC13687BP for the prevention, amelioration or treatment of cognitive dysfunction or Alzheimer's disease.

[0008] [Means for solving the problem] According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cognitive impairment or Alzheimer's disease, comprising Lactobacillus fermentum SRK414 strain, accession number KCTC13687BP, as an active ingredient.

[0009] According to another aspect of the present invention, there is provided a food composition for preventing or ameliorating cognitive impairment or Alzheimer's disease, comprising the Lactobacillus fermentum SRK414 strain, accession number KCTC13687BP, as an active ingredient.

[0010] The present inventors have newly isolated the Lactobacillus fermentum SRK414 strain (Accession Number: KCTC13687BP) and administered fermented milk produced by this strain to an experimental animal model in which secondary osteoporosis was induced, and confirmed that it had the effect of increasing bone mineral density and bone mass ratio (Korean Patent Registered Publication No. 10-2120479 (Announced on June 9, 2020)).

[0011] In the present invention, the Lactobacillus fermentum SRK414 strain was deposited at the Korean Collection for Type Culture (KCTC) of the Korea Institute of Bioscience and Biotechnology under the accession number KCTC13687BP.

[0012] The bacterial properties of the Lactobacillus fermentum SRK414 strain are as follows:

[0013] - containing the 16s rRNA sequence of SEQ ID NO: 1

[0014] - Gram-positive bacillus or cocci in form

[0015] - It is part of the normal flora of the human mouth, gastrointestinal tract, and female genital tract and is generally considered safe for use in food (probiotics and fermented foods). L. fermentum is a member of the genus Lactobacillus, and species within this genus are known for a variety of applications, including food and feed fermentation. Some strains of L. fermentum have been found to be naturally resistant to antibiotics and chemotherapeutic agents.

[0016] In one embodiment of the present invention, the cognitive impairment may be accompanied by symptoms of memory impairment, attention impairment, spatial perception impairment, language impairment, or a combination thereof.

[0017] In one embodiment of the present invention, the cognitive impairment is an amyloid beta accumulation disease selected from the group consisting of, but not limited to, Alzheimer's disease, Parkinson's disease dementia, Lewy body dementia, Huntington's disease dementia, preclinical Alzheimer's disease, and Down's syndrome.

[0018] In another embodiment of the present invention, the cognitive dysfunction is a tau accumulation disease selected from the group consisting of, but not limited to, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), Pick's disease, and frontotemporal dementia (FTD).

[0019] In one embodiment of the present invention, the Lactobacillus fermentum SRK414 strain inhibits the production of amyloid beta protein, tau protein, or a combination thereof in brain tissue.

[0020] In one embodiment of the present invention, the amyloid beta protein is a soluble or insoluble amyloid beta protein.

[0021] In one embodiment of the present invention, the amyloid beta protein is Aβ 40- , Aβ 42- or combinations thereof, but are not limited to these.

[0022] In one embodiment of the present invention, the tau protein is phosphorylated tau protein.

[0023] In one embodiment of the present invention, the tau protein may be, but is not limited to, Tau13, phosphorylated tau (Thr231), phospho-Tau (Ser202, Thr205), phospho-Tau (Thr181), phospho-Tau (Thr212, Ser214), phospho-Tau (Ser396), or phospho-Tau (Ser422).

[0024] In one embodiment of the present invention, the tau protein is a soluble or insoluble tau protein.

[0025] In one embodiment of the present invention, the composition has the effect of reducing barrier permeability compared to a control group not treated with the bacterial strain.

[0026] In one embodiment of the present invention, the composition exhibits an increase in strains of Eubacterium xylanophilum, Ruminococcaceae, Alloprevotella, or a combination thereof in the intestinal microbial flora compared to a control group not treated with the strains.

[0027] In one embodiment of the present invention, the composition exhibits a reduction in strains of Peptococcaceae, Lachnospiraceae NK4A136 group, Clostridia, Staphylococcus, or a combination thereof in the intestinal microbial flora compared to a control group not treated with the bacterial strains.

[0028] The strains that are active ingredients in the compositions of the present invention include isolated and / or purified bacterial cells of the strains, as well as cultures containing bacterial cells, disrupted bacterial cells, extracts of bacterial cells, culture supernatants, concentrates, concentrates, dried products, and, if necessary, diluted solutions or dilutions thereof, and include any state obtained by processing the above-mentioned culture solutions and cultures.

[0029] The method for culturing, extracting, isolating, concentrating, drying, diluting, etc. the bacterial cells is not particularly limited.

[0030] The medium for culturing the fungus usually contains milk proteins such as skim milk, whey, and casein, sugars, yeast extract, etc., and various common aerobic or anaerobic culture methods can be appropriately used.

[0031] The culture temperature is set to, for example, 35°C to 45°C, and a neutral culture method can also be used in which an alkali such as sodium hydroxide is used during culture to maintain the pH of the medium at a neutral to acidic range, for example, a pH of about 5 to 6. In addition to such a neutral culture method, any culture method such as a batch culture method can also be used, and after culture, the culture or its supernatant may be concentrated, dried, diluted, etc., as necessary.

[0032] Alternatively, the supernatant of the culture may be separated from the bacterial cells by centrifugation or membrane separation, and the bacterial cells may be collected in a concentrated state. The bacterial cells may then be subjected to ultrasonic treatment or enzyme treatment to extract intracellular components, or the culture, its supernatant, bacterial cells, or extracts thereof may be dried. These may be used as active ingredients in the composition of the present invention.

[0033] When the composition of the present invention is prepared as a food composition, active ingredients may include not only the lactic acid bacteria but also ingredients commonly added during food production. The added ingredients include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. Examples of carbohydrates that can be used include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, oligosaccharides, etc.), polysaccharides (e.g., common sugars such as dextrin and cyclodextrin), and sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of flavorings that can be used include natural flavorings (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)), and synthetic flavorings (saccharin, aspartame, etc.).

[0034] For example, when the food composition of the present invention is manufactured as a drink, it may further contain, in addition to the strain of bacteria which is the active ingredient of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract, or licorice extract.

[0035] The food compositions of the present invention include processed forms of all natural ingredients, such as foods, functional foods, nutritional supplements, health foods, and food additives. The food compositions of the above types may be prepared in various forms by conventional methods known in the art.

[0036] For example, health foods may be prepared by adding the lactic acid bacteria itself in the form of tea, juice, or drinkable preparations, or by granulating, encapsulating, or powdering the lactic acid bacteria. Foods may also be prepared by adding the extract of the present invention to beverages (including alcoholic beverages), fruits and processed fruits (e.g., canned fruits, bottled fruits, jams, marmalade, etc.), fish, meat, and processed fruits (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, candy, dairy products (e.g., yogurt, fermented milk, butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., miso paste, soy sauce, sauces, etc.). To use the extract of the present invention as a food additive, it can be prepared in the form of a powder or concentrate.

[0037] When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention is one commonly used in formulation, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to these ingredients, the pharmaceutical composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0038] The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably administered orally. The pharmaceutical composition of the present invention can be formulated into various oral or parenteral dosage forms, including, but not limited to, the following:

[0039] Dosage forms for oral administration include, for example, tablets, pills, hard / soft capsules, liquids, suspensions, emulsions, syrups, granules, and elixirs, and these dosage forms may contain, in addition to the active ingredient, one or more commonly used diluents or excipients such as fillers, extenders, wetting agents, disintegrants, lubricants, binders, surfactants, etc. Disintegrants may include agar, starch, alginic acid or its sodium salt, anhydrous calcium hydrogen phosphate, etc. Lubricants may include silica, talc, stearic acid or its magnesium or calcium salt, polyethylene glycol, etc. Binders may include magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low-molecular-weight hydroxypropyl cellulose, etc. Other diluents that can be used include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc., and, in some cases, commonly known boiling mixtures, absorbents, coloring agents, flavoring agents, sweetening agents, etc. can be used together.

[0040] The compositions may be sterilized or may contain preservatives, stabilizers, wetting or emulsifying agents, salts for regulating osmotic pressure, auxiliary substances such as buffers, and other therapeutically useful substances, and can be formulated by conventional mixing, granulating, or coating methods.

[0041] The appropriate dosage of the pharmaceutical composition of the present invention may be variously formulated depending on factors such as the formulation method, administration method, age, weight, sex, pathological condition, diet of the patient, administration time, administration route, excretion rate, and reaction sensitivity.

[0042] The pharmaceutical compositions of the present invention may be prepared in unit dose form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients by a method readily practiced by those skilled in the art to which this invention pertains, and may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may further contain a dispersing agent or stabilizer.

[0043] According to yet another aspect of the present invention, there is provided a method for preventing, ameliorating, or treating cognitive impairment or Alzheimer's disease, comprising administering to a subject the Lactobacillus fermentum SRK414 strain deposited under accession number KCTC13687BP.

[0044] In the present invention, the term "subject" refers to a subject in need of prevention, improvement, or treatment of cognitive impairment or Alzheimer's disease, but is not limited thereto. Specifically, the subject may be a subject who exhibits symptoms of memory impairment, attention impairment, spatial perception ability, language impairment, or a combination thereof, or who has been diagnosed with Alzheimer's disease.

[0045] According to yet another aspect of the present invention, there is provided a method for preventing or treating cognitive impairment or Alzheimer's disease, comprising administering to a subject a pharmaceutical composition comprising, as an active ingredient, Lactobacillus fermentum SRK414 strain having accession number KCTC13687BP.

[0046] According to yet another aspect of the present invention, there is provided a method for preventing or ameliorating cognitive impairment or Alzheimer's disease, the method comprising administering to a subject a food composition containing, as an active ingredient, Lactobacillus fermentum SRK414 strain having accession number KCTC13687BP.

[0047] [Effects of the invention] The present invention provides a composition for preventing, ameliorating, or treating cognitive impairment and Alzheimer's disease, comprising the Lactobacillus fermentum SRK414 strain. The strain of the present invention has the effect of reducing barrier permeability, is highly effective in reducing amyloid beta and tau protein, and is highly effective in improving cognitive function, and can therefore be useful as a food or therapeutic agent for the same purposes.

[0048] [Brief description of the drawing] 1 and 2 show the results of analyzing the intestinal microbial flora after administering the SRK414 strain of the present invention to an animal model of Alzheimer's disease.

[0049] FIG. 3 is a graph showing the barrier permeability of experimental animals after administering the SRK414 strain of the present invention to an Alzheimer's disease animal model.

[0050] FIG. 4 shows the results of amyloid beta immunostaining of brain tissue after administration of the SRK414 strain of the present invention to an Alzheimer's disease animal model.

[0051] FIG. 5 shows the results of ELISA for amyloid beta in brain tissue after administration of the SRK414 strain of the present invention to an animal model of Alzheimer's disease.

[0052] 6 and 7 show the results of Western blot analysis of tau protein in brain tissue after administration of the SRK414 strain of the present invention to an Alzheimer's disease animal model.

[0053] FIG. 8 shows the effect of administering the SRK414 strain of the present invention to an Alzheimer's disease animal model, followed by improving the short-term memory ability and spatial perception ability of the experimental animals in a Y-maze test.

[0054] [Mode for Carrying Out the Invention]

[0055] The present invention will be described in more detail below using examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail, and that the scope of the present invention is not limited to these examples according to the gist of the present invention.

[0056] Example

[0057] Throughout this specification, "%" used to indicate the concentration of a particular substance is (wt / wt)% for solid / solid, (wt / vol)% for solid / liquid, and (vol / vol)% for liquid / liquid, unless otherwise specified.

[0058] Example 1. Probiotics Production

[0059] The Lactobacillus fermentum strain SRK414 used in this experiment was isolated from infant feces. Lactic acid bacteria seed culture was grown in a flask containing MRS broth at 37°C for 24 hours. The seed culture was inoculated into an optimized proprietary medium. Culture was maintained at a constant pH of 5.5-6.0 and stirred at 55-65 RPM for 18-20 hours at 37°C. 40X concentrated cells were lyophilized according to the proprietary manual. After lyophilization, the colony-forming units (CFU) per gram of each probiotic powder were measured by serial dilution. The probiotics were suspended in 1X PBS and diluted to 5 x 10 before use. 9 The density was adjusted to CFU / 200 μL.

[0060] Example 2. Preparation of Alzheimer's disease animal model and experimental group setting

[0061] A transgenic animal model of ADLP (Alzheimer's disease-like pathology) was constructed by crossbreeding i) transgenic 5XFAD mice (Tg6977, Jackson Laboratory, Stock #006554) expressing human APP with Swedish (K670N / M671L), Florida (I716V), and London (V717I) mutations under the control of the Thy1 promoter, ii) transgenic 5XFAD mice expressing human PSEN1 with M146L and L286V mutations, and iii) transgenic JNPL3 mice expressing human Tau with P301L mutation under the control of the prion protein promoter (TauP301L-JNPL3, Taconic, Stock #2508 homozygote).

[0062] ADLP expressing a total of three mutant human genes APT All mice used in this experiment were female, as pathological changes and cognitive impairment associated with Alzheimer's disease appear earlier in female mice than in male mice.

[0063] To confirm the therapeutic effect of the strain SRK414 of the present invention, ADLP mice aged 2 to 2.5 months were cultured. APT Mice were administered 200 μL of either the PBS suspension of strain SRK414 (SRK414) or PBS (NC), and the normal model ADLP WT Mice were orally administered 200 μL of PBS (WT) five times a week for five months.

[0064] Example 3. Gut microbiota evaluation

[0065] To assess the gut microbiota of experimental animals, fecal samples were collected before sacrifice and stored at -80°C until analysis. Genomic DNA was extracted from the fecal samples using the QIAamp DNA Fecal Mini Kit (Qiagen, 51304). The V3-V4 region of the 16s rRNA gene was amplified by PCR using the 341F primer (5'-CCTACGGGNGGCWGCAG-3', SEQ ID NO: 2) and the 805R primer (5'-GACTACHVGGGTATCTAATCC-3', SEQ ID NO: 3). The amplified PCR product was purified using HiAccuBeads (AccuGene, ACN01.50). Metagenomic sequencing was performed using an Ion Torrent S5 sequencer system.

[0066] The generated sequences were used to confirm the overall genetic information of the gut microbiota using the QIIME2 (version 2022.2) pipeline. To identify differences in microbial genera between the SRK414-treated and control groups, the linear discriminant analysis (LDA) effect size (LEfSe) algorithm was used at the genus level.

[0067] The results are shown in Figures 1 and 2.

[0068] As shown in Figures 1 and 2, in the SRK414-treated group, Eubacterium xylanophilum group, Ruminococcaceae, Alloprevotella, etc. were up-regulated, while Peptococcaceae, Lachnospiraceae NK4A136 group, Clostridia, Staphylococcus, etc. were down-regulated.

[0069] Example 4. Barrier permeability evaluation

[0070] A 4kDa fluorescent substance was used to assess the degree of barrier permeability in experimental animals. After fasting for 4 hours, the 4kDa fluorescent substance (0.6 mg / g body weight) was orally administered. Two hours after administration, the substance in the intestine was imaged and the residual amount was measured to assess the barrier permeability.

[0071] The results are shown in Figure 3.

[0072] As shown in Figure 3, ADLP APT The mice had increased barrier permeability compared to normal mice, and two hours after administration of the fluorescent substance, the amount of fluorescent substance remaining in the intestinal tract was significantly reduced. On the other hand, in the group administered with the SRK414 strain, the amount of fluorescent substance remaining in the intestinal tract was increased two hours after administration compared to the NC group, confirming that the SRK414 strain reduced barrier permeability.

[0073] Example 5. Aβ immunostaining (immunohistochemistry) and ELISA (enzyme-linked immunosorbent assay)

[0074] 5-1.Immunostaining

[0075] Experimental animals were anesthetized with a mixture of tiletamine-zolazepam and xylazine (1.2 mg / kg) and perfused with PBS. For immunofluorescence staining, brain tissue from experimental animals was fixed in 4% paraformaldehyde for 24 hours and dehydrated in 30% sucrose solution for approximately 72 hours. The pretreated brain tissue was frozen at -80°C and then cut into coronal sections using a microtome capable of maintaining a temperature of -25°C. After washing with PBS, the brain tissue sections underwent blocking and permeabilization, and then reacted with biotin-conjugated primary antibodies: 4G8 (1:700, Covance, SIG-39240), GFAP (1:1,000, Invitrogen, 13-0300), and Iba1 (1:500, Wako, 019-19741). The brain tissue sections reacted with the primary antibody were washed with PBS, then further reacted with a fluorescently labeled secondary antibody, and then observed under a confocal microscope.

[0076] The results are shown in Figure 4.

[0077] As shown in Figure 4, the area occupied by Aβ plaques in the brain was significantly reduced in the group administered with the inventive strain SRK414 compared to the NC group, confirming that the strain SRK414 alleviates Aβ pathology in the brain.

[0078] 5-2. Human Aβ ELISA

[0079] Experimental animals were anesthetized with a mixture of tiletamine-zolazepam and xylazine (1.2 mg / kg) and perfused with PBS. For ELISA experiments, brain tissue from experimental animals was homogenized in RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Nonidet P-40; 0.1% SDS; 0.5% deoxycholate sodium salt), and the protein concentration in the supernatant of the tissue extract was quantified. 100 μg of protein was separated into RIPA-soluble and RIPA-insoluble fractions by ultracentrifugation. The RIPA-insoluble fraction was resuspended in 70% formic acid and neutralized with 1 M Tris-base solution before ELISA experiments. RIPA-soluble Aβ 40 and Aβ 42 and RIPA-insoluble Aβ 40 and Aβ 42-- The concentration of was measured according to the protocol of the human Aβ-specific ELISA kit.

[0080] The results are shown in Figure 5.

[0081] As shown in FIG. 5, in the group administered with the strain SRK414 of the present invention, the soluble Aβ in the brain 42- was significantly reduced compared to the NC group, and soluble Aβ 40- , insoluble Aβ 40- and Aβ 42- showed a tendency to decrease, confirming that strain SRK414 alleviates Aβ pathology in the brain.

[0082] Example 6. Tau Western blotting

[0083] 6-1. Sarkosyl-insoluble fractionation

[0084] Experimental animals were anesthetized with a mixture of tiletamine-zolazepam and xylazine (1.2 mg / kg) and perfused with PBS. Brain tissue from experimental animals was homogenized in Tris buffer (TBS; 150 mM NaCl; 25 mM Tris-HCl, pH 7.4; 1 mM EDTA; 1 mM EGTA). The supernatant of the tissue extract was then reacted with a 1% N-lauroyl sarcosine sodium salt solution at 37°C for 1 hour. After the reaction, the sample was ultracentrifuged to separate the sarkosyl-soluble and sarkosyl-insoluble fractions.

[0085] 6-2. Western blotting

[0086] The sarkosyl-soluble fraction was subjected to Western blotting after protein quantification. The sarkosyl-insoluble fraction was resuspended in 5X sample buffer and boiled at 70°C for 10 minutes before Western blotting. Proteins in the sample were separated by SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis) and then transferred to a PVDF (polyvinylidene fluoride) membrane. For immunoblotting, the membrane was blocked with 5% skim milk and then incubated at 4°C with primary antibodies: APP (6E10, 1:1,000, Covance, SIG-39320), Tau13 (1:2,000, Abcam, ab13090), phospho-Tau (Thr231; AT180, 1:1,000, Invitrogen, MN1040), and phospho-Tau. The cells were incubated with phospho-Tau (Ser202, Thr205; AT8, 1:1,000, Invitrogen, MN1020), phospho-Tau (Thr181; AT270, 1:1,000, Invitrogen, MN1050), phospho-Tau (Thr212, Ser214; AT100, 1:1,000, Invitrogen, MN1060), phospho-Tau (Ser396, 1:2,000, Thermo Fisher Scientific, 44-752G), phospho-Tau (Ser422, 1:1,000, Thermo Fisher Scientific, 44-764ZG), and β-actin (1:2,000, Cell Signaling Technology, #3700) for over 16 hours. After washing with TBST (Tris-buffered saline with 0.05% Tween 20), the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. The membrane was then reacted with enhanced chemiluminescent substrate (ECL) (Abfrontier, LF-QC0103) and detected using an image analyzer (LAS-3000; Fujifilm Corporation).The intensity of each band on the membrane was quantified using Multigauge software (Fujifilm Corporation).

[0087] The results are shown in FIGS.

[0088] As shown in Figures 6 and 7, in the group administered with the strain SRK414 of the present invention, phosphorylated Tau Ser396 was significantly reduced compared to the NC group, and insoluble Tau13, phosphorylated Tau Ser422, Ser202, and Thr205 tended to decrease.

[0089] Example 7. Y-maze test

[0090] A Y-maze test was conducted to evaluate the short-term memory and spatial perception abilities of experimental animals. One day before the test, the experimental animals were adapted to the test room and maze apparatus. On the test day, the experimental animals were placed in the center of the Y-maze and allowed to explore all three arms of the maze apparatus for 8 minutes. The total number of entries was defined as the number of times the experimental animals entered an arm, and the number of spontaneous alternations was defined as the number of times the experimental animals entered three different arms consecutively. The alternation ratio was calculated as the number of alternations / number of possible alternations (total number of entries - 2) × 100.

[0091] The results are shown in Figure 8.

[0092] As shown in Figure 8, the alternation rate in the group administered with the present strain SRK414 was significantly increased compared to the NC group, confirming that the strain SRK414 has the effect of improving short-term memory ability and spatial perception ability.

[0093] [Table 1]

[0094] [Accession number]

[0095] Depository institution: Korea Center for Biological Resources (KCTC)

[0096] Accession number: KCTC13687BP

[0097] Date of acceptance: 20181025 [Brief explanation of the drawings]

[0098] [Figure 1] 1 shows the results of analyzing the intestinal microbial flora after administering the SRK414 strain of the present invention to an Alzheimer's disease animal model. [Figure 2] 1 shows the results of analyzing the intestinal microbial flora after administering the SRK414 strain of the present invention to an Alzheimer's disease animal model. [Figure 3] FIG. 1 shows the barrier permeability of experimental animals after administering the SRK414 strain of the present invention to an Alzheimer's disease animal model. [Figure 4] 1 shows the results of amyloid beta immunostaining of brain tissue after administration of the SRK414 strain of the present invention to an Alzheimer's disease animal model. [Figure 5] 1 shows the results of ELISA for amyloid beta in brain tissue after administering the SRK414 strain of the present invention to an Alzheimer's disease animal model. [Figure 6] 1 shows the results of Western blot of tau protein in brain tissue after administration of the SRK414 strain of the present invention to an Alzheimer's disease animal model. [Figure 7] 1 shows the results of Western blot of tau protein in brain tissue after administration of the SRK414 strain of the present invention to an Alzheimer's disease animal model. [Figure 8] FIG. 1 shows the effect of administering the SRK414 strain of the present invention to an Alzheimer's disease animal model on improving short-term memory ability and spatial perception ability in a Y-maze test.

Claims

1. A pharmaceutical composition for preventing or treating cognitive impairment or Alzheimer's disease, comprising Lactobacillus fermentum SRK414 strain, accession number KCTC13687BP, as an active ingredient.

2. The pharmaceutical composition of claim 1, wherein the cognitive impairment is accompanied by symptoms of memory impairment, attention impairment, spatial perception impairment, language impairment, or a combination thereof.

3. The pharmaceutical composition according to claim 1, wherein the Lactobacillus fermentum SRK414 strain inhibits the production of amyloid beta protein, tau protein, or a combination thereof in brain tissue.

4. The pharmaceutical composition of claim 1, wherein the amyloid beta protein is a soluble or insoluble amyloid beta protein.

5. The pharmaceutical composition of claim 1 , wherein the tau protein is a phosphorylated tau protein.

6. The pharmaceutical composition of claim 1, wherein the composition has the effect of reducing barrier permeability compared to a control group not treated with the bacterial strain.

7. The pharmaceutical composition of claim 1, wherein the composition exhibits an increase in strains belonging to Eubacterium xylanophilum, Ruminococcaeae, Alloprevotella, or a combination thereof in the intestinal microbial flora compared to a control group not treated with the bacterial strains.

8. 2. The pharmaceutical composition of claim 1, wherein the composition exhibits a reduction in strains belonging to the Peptococcus, Lachnospiraceae NK4A136 group, Clostridia, Staphylococcus, or a combination thereof in the intestinal microbial flora compared to a control group not treated with the bacterial strains.

9. A food composition for preventing or ameliorating cognitive impairment or Alzheimer's disease, comprising the Lactobacillus fermentum SRK414 strain, accession number KCTC13687BP, as an active ingredient.

Images